Dominant-negative mutants of human MxA protein: domains in the carboxy-terminal moiety are important for oligomerization and antiviral activity.

Human MxA protein is an interferon-induced 76-kDa GTPase that exhibits antiviral activity against several RNA viruses. Wild-type MxA accumulates in the cytoplasm of cells. TMxA, a modified form of wild-type MxA carrying a foreign nuclear localization signal, accumulates in the cell nucleus. Here we show that MxA protein is translocated into the nucleus together with TMxA when both proteins are expressed simultaneously in the same cell, demonstrating that MxA molecules form tight complexes in living cells. To define domains important for MxA-MxA interaction and antiviral function in vivo, we expressed mutant forms of MxA together with wild-type MxA or TMxA in appropriate cells and analyzed subcellular localization and interfering effects. An MxA deletion mutant, MxA(359-572), formed heterooligomers with TMxA and was translocated to the nucleus, indicating that the region between amino acid positions 359 and 572 contains an interaction domain which is critical for oligomerization of MxA proteins. Mutant T103A with threonine at position 103 replaced by alanine had lost both GTPase and antiviral activities. T103A exhibited a dominant-interfering effect on the antiviral activity of wild-type MxA rendering MxA-expressing cells susceptible to infection with influenza A virus, Thogoto virus, and vesicular stomatitis virus. To determine which sequences are critical for the dominant-negative effect of T103A, we expressed truncated forms of T103A together with wild-type protein. A C-terminal deletion mutant lacking the last 90 amino acids had lost interfering capacity, indicating that an intact C terminus was required. Surprisingly, a truncated version of MxA representing only the C-terminal half of the molecule exerted also a dominant-negative effect on wild-type function, demonstrating that sequences in the C-terminal moiety of MxA are necessary and sufficient for interference. However, all MxA mutants formed hetero-oligomers with TMxA and were translocated to the nucleus, indicating that physical interaction alone is not sufficient for disturbing wild-type function. We propose that dominant-negative mutants directly influence wild-type activity within hetero-oligomers or else compete with wild-type MxA for a cellular or viral target.     

Expression of the antiviral protein MxA in cells transiently perturbs endocytosis

MxA is an interferon-induced antiviral protein. Viral replication relies on the trafficking machinery of the host cell. Overexpression of MxA was found to perturb trafficking of internalized transferrin resulting in its accumulation in cells. Interestingly, this perturbation of endocytic trafficking was transient--with a maximal effect being seen 5-6 h after transfection. By 12 h after transfection the perturbation of endocytosis was seen to have subsided although MxA protein levels remained elevated even 24 h after transfection. The accumulation of transferrin is due to a block in transferrin recycling. It is further shown that MxA can physically associate with the endocytic protein dynamin, possibly accounting for the observed effect of MxA expression on transferrin endocytosis. These results uncover a hitherto unknown aspect of MxA action on trafficking processes within cells.     

A monomeric GTPase-negative MxA mutant with antiviral activity

MxA is a large, interferon-induced GTPase with antiviral activity against RNA viruses. It forms large oligomers, but whether oligomerization and GTPase activity are important for antiviral function is not known. The mutant protein MxA(L612K) carries a lysine-for-leucine substitution at position 612 and fails to form oligomers. Here we show that monomeric MxA(L612K) lacks detectable GTPase activity but is capable of inhibiting Thogoto virus in transiently transfected Vero cells or in a Thogoto virus minireplicon system. Likewise, MxA(L612K) inhibited vesicular stomatitis virus multiplication. These findings indicate that MxA monomers are antivirally active and suggest that GTP hydrolysis may not be required for antiviral activity. MxA(L612K) is rapidly degraded in cells, whereas wild-type MxA is stable. We propose that high-molecular-weight MxA oligomers represent a stable intracellular pool from which active MxA monomers are recruited.     

Interferon-induced antiviral protein MxA interacts with the cellular RNA helicases UAP56 and URH49

Mx proteins are a family of large GTPases that are induced exclusively by interferon-α/β and have a broad antiviral activity against several viruses, including influenza A virus (IAV). Although the antiviral activities of mouse Mx1 and human MxA have been studied extensively, the molecular mechanism of action remains largely unsolved. Because no direct interaction between Mx proteins and IAV proteins or RNA had been demonstrated so far, we addressed the question of whether Mx protein would interact with cellular proteins required for efficient replication of IAV. Immunoprecipitation of MxA revealed its association with two closely related RNA helicases, UAP56 and URH49. UAP56 and its paralog URH49 play an important role in IAV replication and are involved in nuclear export of IAV mRNAs and prevention of dsRNA accumulation in infected cells. In vitro binding assays with purified recombinant proteins revealed that MxA formed a direct complex with the RNA helicases. In addition, recombinant mouse Mx1 was also able to bind to UAP56 or URH49. Furthermore, the complex formation between cytoplasmic MxA and UAP56 or URH49 occurred in the perinuclear region, whereas nuclear Mx1 interacted with UAP56 or URH49 in distinct dots in the nucleus. Taken together, our data reveal that Mx proteins exerting antiviral activity can directly bind to the two cellular DExD/H box RNA helicases UAP56 and URH49. Moreover, the observed subcellular localization of the Mx-RNA helicase complexes coincides with the subcellular localization, where human MxA and mouse Mx1 proteins act antivirally. On the basis of these data, we propose that Mx proteins exert their antiviral activity against IAV by interfering with the function of the RNA helicases UAP56 and URH49.     

Specificities of RNA N-glycosidase activity of Mirabilis antiviral protein variants.

Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, inactivates both eukaryotic and prokaryotic ribosomes by means of site-specific RNA N-glycosidase activity. In order to identify the site of this activity, some amino acid residues of MAP, conserved in homologous ribosome-inactivating proteins, were altered to other amino acids by replacing DNA fragments of the total synthetic gene of MAP. When the in vitro proteins synthesis of rabbit reticulocyte was treated with MAP variants secreted into culture media of Escherichia coli transformants, the inhibitory effect of R26L and R48L (R26L designates MAP variant with Arg-26 changed to Leu) was found to be similar to that of native MAP. Both purified Y72F and Y118F had the same effect as native MAP, and E168D had a slightly weaker effect. In contrast, on the protein synthesis of E. coli, Y118F had one-tenth the effect of native MAP, and Y72F and E168D approximately one-hundredth the effect. These three variant proteins also exhibited reduced RNA N-glycosidase activity on substrate E. coli ribosomes. These results suggest that Tyr-72 and Glu-168 are involved in RNA N-glycosidase activity. When the R171K gene was expressed in E. coli, an N-glycosidic bond of the 23 S rRNA of the host ribosome was found to be cleaved, although no product of the gene could be detected. This suggests that MAP variants can maintain their N-glycosidase activity when the conserved Glu-168 and Arg-171 are changed to similarly charged residues.     

Nonspecificity of antiviral protein action]

The authors studied the antiviral activity of the informative RNA of antiviral protein (M-RNA AVP) isolated from the cells following superinduction of interferon in them for the purpose of ascertaining the action specificity of the product of their translation--AVP. Following the administration of M-RAN AVP a marked (from 1 to 5 lg PFU/mil) reduction of the infectious titres was observed in the homologous and heterologous cells. RNA preparations from control (noninduced) cells possessed a weak (0.4--0.1 lg PFU/mil) antiviral activity.     

Enhanced virus resistance of transgenic mice expressing the human MxA protein.

MxA is a GTPase that accumulates to high levels in the cytoplasm of interferon-treated human cells. Expression of MxA cDNA confers to transfected cell lines a high degree of resistance against several RNA viruses, including influenza, measles, vesicular stomatitis, and Thogoto viruses. We have now generated transgenic mice that express MxA cDNA in the brain and other organs under the control of a constitutive promoter. Embryonic fibroblasts derived from the transgenic mice were nonpermissive for Thogoto virus and showed reduced susceptibility for influenza A and vesicular stomatitis viruses. The transgenic animals survived challenges with high doses of Thogoto virus by the intracerebral or intraperitoneal route. Furthermore, the transgenic mice were more resistant than their nontransgenic littermates to intracerebral infections with influenza A and vesicular stomatitis viruses. These results demonstrate that MxA is a powerful antiviral agent in vivo, indicating that it may protect humans from the deleterious effects of infections with certain viral pathogens.     

Mechanism of interferon action. II. Induction and decay kinetics of the antiviral state and protein P54 in human amnion U cells treated with gamma interferon

The kinetics of induction and decay of the antiviral state and polypeptide p54 expression induced by recombinant human interferon gamma (rIFN-gamma) were examined in human amnion U cells. The kinetics of induction of the antiviral state, as measured by the single-cycle yield reduction of vesicular stomatitis virus, were first order over the period of about 6-12 h following a lag of about 2-4 h. The induction of p54 synthesis by rIFN-gamma slightly preceded the induction of the antiviral state. The kinetics of p54 induction were first order over a period of about 2-8 h after a lag of about 1 h. The rate of polypeptide p54 synthesis induced by rIFN-gamma decayed significantly within 1 day after the removal of IFN. However, polypeptide p54 was comparatively stable, displaying a half-life of about 3 days. The antiviral state likewise decayed significantly within 3-4 days following removal of IFN-gamma, and by 5-8 days, the virus yields were comparable to those of untreated control cell cultures. These results suggest that polypeptide p54 may play an important role in the antiviral action of rIFN-gamma in human amnion U cells.     

Expression of human MxA protein in mosquito cells interferes with LaCrosse virus replication

Human MxA protein inhibits LaCrosse virus (LAC virus; family Bunyaviridae) replication in vertebrate cells and MxA-transgenic mice. LAC virus is transmitted to humans by Aedes triseriatus mosquitoes. In this report, we have shown that transfected mosquito cells expressing the human MxA cDNA are resistant to LAC virus but permissive for Sindbis virus (family Togaviridae) infection.     

5-Formyl-2'-deoxyuridine: cytostatic and antiviral properties and possible modes of action.

5-Formyl-2'-deoxyuridine (fdUrd) was prepared by a new method starting from thymidine and investigated for its influence both on proliferation of cultured mammalian cells and virus replication in vitro. The compound was found to have strong cytostatic and antiviral properties: 50% inhibition of proliferation of BHK 21/C13 cells or Ehrlich ascites tumour cells (EAT) was obtained at 4 - 10(-6) and 6 - 10(-6) M, respectively, while the treatment of pseudorabies virus with the same concentration resulted in about 1.5 log reduction of virus yield. A concentration of 1 - 10(-4) M inhibited cell proliferation by 80 to 100% while the virus yield was reduced by more than 3 orders of magnitude. All inhibitions can be prevented by thymidine.--DNA synthesis of EAT cells in vitro, as estimated by incorporation of [32P]-phosphate or low concentrations of [3H]-thymidine, was inhibited. Further biochemical experiments have provided indirect evidence that the compound is phosphorylated by thymidine and thymidylate phosphorylating enzymes. An inhibition of cell free DNA synthesis was found to be depending on a given period of preincubation with the compound (supposed to be needed for the formation of fdUrd 5'-triphosphate). This suggests that the 5'-triphosphate of fdUrd is an inhibitor of DNA polymerases and--by analogy with experiments with 5-formyluridine-5'-triphosphate and RNA polymerases [14]--may be used as an affinity label for this group of enzymes. It is concluded that the described cytostatic and antiviral effects of fdUrd are due to an intracellular "lethal" synthesis of the relevant phosphates which inhibit thymidylate synthetase (as had been found earlier to occur with the chemically prepared nucleotide in cell free extracts [1, 2]) and DNA synthesizing enzymes.     

Mechanism of dnaB protein action. I. Crystallization and properties of dnaB protein, an essential replication protein in Escherichia coli

Purification and crystallization of dnaB protein from Escherichia coli was performed on a large scale by a simple procedure. From 1.5 kg of cells, 520 mg of dnaB protein were obtained in a 58% yield with a purity greater than 99%. The E. coli cells harbor a high copy-number plasmid carrying the dnaB gene and overproduce the enzyme over 200-fold. The subunit molecular weight determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is 50,000. Based on a native Mr = 290,000 and cross-linking studies that yielded six bands, dnaB protein is judged to be a hexamer, confirming the results of Reha-Krantz, L. J., and Hurwitz, J. (1978) J. Biol. Chem. 253, 4043-4050.     

Interferon-induced human protein MxA is a GTPase which binds transiently to cellular proteins.

MxA is an abundant and ubiquitous cytoplasmic protein induced by alpha/beta interferon in human cells. Upon full induction, it can constitute 0.5 to 1% of cytosolic proteins. MxA can bind elements of the cytoskeleton, such as actin and tubulins, and several larger cellular proteins. However, these protein-protein interactions seem to be transitory. The human MxA protein contains a tripartite GTP-binding domain consisting of GxxxxGKS, DxxG, and TKxD, where x is any amino acid. It is shown here that the native MxA protein has GTPase activity (GTP----GDP) when purified by immunoprecipitation with affinity-purified polyclonal antibodies directed against the C-terminal domain of MxA. The GTPase activity is greatly diminished by polyclonal antibodies directed against the N-terminal domain of MxA (the domain which contains the GTP-binding consensus elements). Amino acid substitution within the GTP-binding domain abolished the GTPase activity of the mutated MxA protein expressed in transfected CHO cells. The reaction is specific for GTP, and the approximate Km is 0.1 mM. The reaction has an absolute requirement for Mg2+. The turnover number is approximately 70 molecules of GTP hydrolyzed per min per MxA molecule. It is suggested that the human MxA protein has certain characteristics of the stress proteins.     

Mechanism of interferon action. Kinetics of decay of the antiviral state and protein phosphorylation in mouse fibroblasts treated with natural and cloned interferons

The kinetics of decay of the antiviral state and protein phosphorylation induced with natural mouse interferon (IFN) and with cloned human IFN were examined in monolayer cultures of mouse Ll929 fibroblast cells. The antiviral state measured by single cycle virus yield reduction with either vesicular stomatitis virus or reovirus decayed significantly within 2 to 3 days following removal of IFN and by 5 to 8 days virus yields had returned to the level of untreated control cells. Trypsinization of IFN-treated cells did not detectably alter the rate of decay of the antiviral state; however, the decay occurred slightly more rapidly in actively growing as compared to stationary cell cultures. The decay of the IFN-induced protein kinase which catalyzes the phosphorylation of endogenous protein P1 and purified initiation factor eIF-2 alpha correlated with the decay of the antiviral state. The decay rates of the antiviral state and protein kinase observed in mouse L929 cells that had been treated with natural mouse IFN synthesized in Newcastle disease virus-induced L929 cells were comparable to the decay rates observed in L929 cells that had been treated with recombinant human IFN-alpha A/D synthesized in Escherichia coli. The induction and decay of the antiviral state and protein kinase following treatment with a single dose of IFN did not significantly affect the sensitivity of the cell population to a subsequent treatment with a single dose of IFN. However, continuous treatment of L929 cells with natural mouse IFN or recombinant human IFN prevented the decay of both the antiviral state and protein kinase but also ultimately lead to cell death. The results suggest that protein phosphorylation may play an important role in the mechanism of IFN action in mouse L929 fibroblasts.     

Mechanism of interferon action. Kinetics of induction of the antiviral state and protein phosphorylation in mouse fibroblasts treated with natural and cloned interferons

The induction of phosphorylation of both protein P1 and protein synthesis initiation factor eIF-2 alpha and the inhibition of virus replication were examined in mouse L929 fibroblasts treated with either natural mouse or individual cloned human interferons (IFN). Natural mouse IFN synthesized in Newcastle disease virus-induced L929 cells and two cloned human leukocyte IFN subspecies synthesized in Escherichia coli, IFN-alpha D and IFN-alpha A/D, possessed antiviral activity in L929 cells as measured by single cycle virus yield reduction with both vesicular stomatitis virus and reovirus. Natural L929 IFN and cloned IFNs, alpha D and alpha A/D, also induced the protein kinase that catalyzed the phosphorylation of endogenous ribosome-associated protein P1 and the alpha subunit of purified initiation factor eIF-2. Two other cloned human IFNs, alpha A and alpha D/A, were poor inducers of both the antiviral state and the phosphorylation of P1 and eIF-2 alpha in mouse L929 cells. The ability of individual human IFN-alpha subspecies to induce P1 and eIF-2 alpha phosphorylation in mouse L929 cells correlated with their ability to induce an antiviral state. Furthermore, the detailed kinetics of induction, in mouse L929 cells, of P1 and eIF-2 alpha phosphorylation and of the antiviral state by the heterologous cloned human IFN-alpha A/D were equivalent to the kinetics of induction by the homologous natural mouse L929 IFN. These results suggest that different subspecies of biologically active IFN induce equivalent antiviral activities and biochemical changes in mouse L929 cells, and that protein phosphorylation may play a major role in the antiviral mechanism of IFN action in mouse L929 fibroblasts.     

Mechanism of action of sparsomycin in protein synthesis.

Before CI isomerizes to C*I, we detect a competitive phase of inhibition (Ki = k5/k4 = 0.05 microM) which eventually, by increasing the concentration of I, becomes linear mixed noncompetitive and involves C*I in place of CI. The equilibration of C and I according to reaction 2 is much slower than the equilibration between C and S in reaction 1 (time-dependent inhibition). The inactivation plots obey reaction 2 and allow us to estimate k6 as equal to 2.2 min-1. The isomerized C*I, free of excess I, can be studied as a mixture with complex C. From the kinetics of the regeneration of C from C*I, in the presence of puromycin, we can estimate k7 to be between 0.22 min-1 and 0.06 min-1. Although the isomerized C*I survives after adsorption on cellulose nitrate filter disks, it does not survive after gel chromatography on a Sepharose CL-4B column but is converted quantitatively to complex C containing D of unchanged reactivity. This result does not support the proposed [Flynn, G. A., & Ash, R. J., (1990) Biochem. Biophys. Res. Commun. 166, 673-680] chemical reaction between D and I toward new products. The isomerized C*I can be obtained not only from the already-made complex C but also de novo from D, R, and M. In the latter case, the reactions which lead to C are represented by the following hypothetical scheme: D + R + M in equilibrium with DRM or C (binding reaction). When C*I is formed de novo, this reaction is coupled to reaction 2 and the ultimate product is a mixture of C and C*I.(ABSTRACT TRUNCATED AT 250 WORDS)     

Resistance to influenza virus and vesicular stomatitis virus conferred by expression of human MxA protein.

MxA and MxB are interferon-induced proteins of human cells and are related to the murine protein Mx1, which confers selective resistance to influenza virus. In contrast to the nuclear murine protein Mx1, MxA and MxB are located in the cytoplasm, and their role in the interferon-induced antiviral state was unknown. In this report we show that transfected cell lines expressing MxA acquired a high degree of resistance to influenza A virus. Surprisingly, MxA also conferred resistance to vesicular stomatitis virus. Expression of MxA in transfected 3T3 cells had no effect on the multiplication of two picornaviruses, a togavirus, or herpes simplex virus type 1. Treatment of MxA-expressing cells with antibodies to mouse alpha-beta interferon did not abolish the resistance phenotype. The conclusion that resistance to influenza virus and vesicular stomatitis virus was due to the specific action of MxA is further supported by the observation that transfected 3T3 cell lines expressing the related MxB failed to acquire virus resistance.